1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to modification of lignin biosynthesis.
2. Description of the Related Art
Lignin is the major structural component of secondarily thickened plant cell walls. It is a complex polymer of hydroxylated and methoxylated phenylpropane units, linked via oxidative coupling that is probably catalyzed by both peroxidases and laccases (Boudet, et al., 1995. “Tansley review No. 80: Biochemistry and molecular biology of lignification,” New Phytologist 129:203-236). Lignin imparts mechanical strength to stems and trunks, and hydrophobicity to water-conducting vascular elements. Although the basic enzymology of lignin biosynthesis is reasonably well understood, the regulatory steps in lignin biosynthesis and deposition remain to be defined (Davin, L. B. and Lewis, N. G. 1992. “Phenylpropanoid metabolism: biosynthesis of monolignols, lignans and neolignans, lignins and suberins,” Rec Adv Phytochem 26:325-375).
There is considerable interest in the potential for genetic manipulation of lignin levels and/or composition to help improve digestibility of forages and pulping properties of trees (Dixon, et al., 1994. “Genetic manipulation of lignin and phenylpropanoid compounds involved in interactions with microorganisms,” Rec Adv Phytochem 28:153178; Tabe, et al., 1993. “Genetic engineering of grain and pasture legumes for improved nutritive value,” Genetica 90:181-200; Whetten, R. and Sederoff, R. 1991. “Genetic engineering of wood,” Forest Ecology and Management 43:301-316; U.S. Patent Appl. Pub. 2004/0049802.). Small decreases in lignin content have been reported to positively impact the digestibility of forages (Casler, M. D. 1987. “In vitro digestibility of dry matter and cell wall constituents of smooth bromegrass forage,” Crop Sci 27:931-934). By improving the digestibility of forages, higher profitability can be achieved in cattle and related industries. In forestry, chemical treatments necessary for the removal of lignin from trees are costly and potentially polluting.
Lignins contain three major monomer species, termed p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S), produced by reduction of CoA thioesters of coumaric, ferulic and sinapic acids, respectively. In angiosperms, guaiacyl and syringyl units predominate, and the S/G ratio affects the physical properties of the lignin. The S and G units are linked through five different dimer bonding patterns (Davin, L. B. and Lewis, N. G. 1992. Rec Adv Phytochem 26:325-375). The mechanisms that determine the relative proportions of these linkage types in a particular lignin polymer have been unknown. Furthermore, there is considerable debate as to whether lignin composition and structure are tightly controlled, or are flexible depending upon monomer availability (Lewis, N. G. 1999. “A 20th century roller coaster ride: a short account of lignification,” Current Opinion in Plant Biology 2:153-162; Sederoff, et al., 1999, “Unexpected variation in lignin,” Current Opinion in Plant Biology 2:145-152).
Lignin levels increase with progressive maturity in stems of forage crops, including legumes such as alfalfa (Jung, H. G. and Vogel, K. P. 1986. “Influence of lignin on digestibility of forage cell wall material,” J Anim Sci 62:1703-1712) and in grasses such as tall fescue (Buxton, D. R. and Russell, J. R. 1988. “Lignin constituents and cell wall digestibility of grass and legume stems,” Crop Sci 28:553-558). In addition, lignin composition changes with advanced maturity towards a progressively higher S/G ratio (Buxton, D. R. and Russell, J. R. 1988. Crop Sci 28:553-558). Both lignin concentration (Albrecht, et al., 1987. “Cell-wall composition and digestibility of alfalfa stems and leaves,” Crop Sci 27:735-741; Casler, M. D. 1987. Crop Sci 27:931-934; Jung, H. G. and Vogel, K. P. 1986. J Anim Sci 62:1703-1712) and lignin methoxyl content, reflecting increased S/G ratio (Sewalt, et al., 1996. “Lignin impact on fiber degradation. 1. Quinone methide intermediates formed from lignin during in vitro fermentation of corn stover,” J Sci Food Agric 71:195-203), have been reported to negatively correlate with forage digestibility for ruminant animals.
Although a number of studies have linked decreased forage digestibility to increased S/G ratio as a function of increased maturity (Buxton, D. R. and Russell, J. R. 1988. Crop Sci 28:553-558; Grabber, et al., 1992. “Digestion kinetics of parenchyma and sclerenchyma cell walls isolated from orchardgrass and switchgrass,” Crop Sci 32: 806-810), other studies have questioned the effect of lignin composition on digestibility (Grabber, et al., 1997. “p-hydroxyphenyl, guaiacyl, and syringyl lignins have similar inhibitory effects on wall degradability,” J Agric Food Chem 45:2530-2532). Further, the hardwood gymnosperm lignins are highly condensed, essentially lacking S residues, and this makes them less amenable to chemical pulping, in apparent contradiction to the concept that reducing S/G ratio would be beneficial for forage digestibility. The reported lack of agreement in the relationship of lignin composition to forage digestibility and chemical pulping is partly due to the fact that the studies to date either have been in vitro, or have compared plant materials at different developmental stages, different varieties or even different species. Therefore, the development of isogenic lines that can be directly compared to reveal the effects of altered S/G ratio on forage digestibility would be beneficial.
To date, there have been very few published reports on the genetic modification of lignin in forage crops such as alfalfa, other Medicago sp., timothy, bromegrass, white or red clover, fescue, orchardgrass, Lolium sp. (e.g. rye grass), and bluegrass among others. Most studies having concentrated on model systems such as Arabidopsis and tobacco (Hoffmann et al., 2004), or tree species such a poplar, and thus the effect of such modifications on forage digestibility is unclear.
In one study, down-regulation of cinnamnyl alcohol dehydrogenase, an enzyme later in the monolignol pathway than COMT or CCOMT, led to a small but significant improvement in in vitro dry matter digestibility in transgenic alfalfa (Baucher, et al., 1999. Plant Mol Biol 39:437-447). U.S. Pat. No. 5,451,514 discloses a method of altering the content or composition of lignin in a plant by stably incorporating into the genome of the plant a recombinant DNA encoding an mRNA having sequence similarity to cinnamyl alcohol dehydrogenase. U.S. Pat. No. 5,850,020 discloses a method for modulating lignin content or composition by transforming a plant cell with a DNA construct with at least one open reading frame coding for a functional portion of one of several enzymes isolated from Pinus radiata (pine) or a sequence having 99% homology to the isolated gene: cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyltransferase (OMT), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL), and peroxidase (POX). U.S. Pat. No. 5,922,928 discloses a method of transforming and regenerating Populus species to alter the lignin content and composition using an O-methyltransferase gene. U.S. Pat. No. 6,610,908 describes manipulation of lignin composition in plants using a tissue-specific promoter and a sequence encoding a ferulate-5-hydroxylase (F5H) enzyme.
While the foregoing studies have provided a further understanding of the production of plant lignin, there remains a great need in the art for plants with greatly improved digestibility as a result of lignin modification. Development of such plants would have a significant benefit in agriculture, particularly for the production of improved forage crops and more particularly forage legumes.